Focus: A Multifaceted Battle Against Cancer: Extracting Knowledge from Chemical Imaging Data Using Computational Algorithms for Digital Cancer Diagnosis

Fourier transform infrared (FTIR) spectroscopic imaging is an emerging microscopy modality for clinical histopathologic diagnoses as well as for biomedical research. Spectral data recorded in this modality are indicative of the underlying, spatially resolved biochemical composition but need computerized algorithms to digitally recognize and transform this information to a diagnostic tool to identify cancer or other physiologic conditions. Statistical pattern recognition forms the backbone of these recognition protocols and can be used for highly accurate results. Aided by biochemical correlations with normal and diseased states and the power of modern computer-aided pattern recognition, this approach is capable of combating many standing questions of traditional histology-based diagnosis models. For example, a simple diagnostic test can be developed to determine cell types in tissue. As a more advanced application, IR spectral data can be integrated with patient information to predict risk of cancer, providing a potential road to precision medicine and personalized care in cancer treatment. The IR imaging approach can be implemented to complement conventional diagnoses, as the samples remain unperturbed and are not destroyed. Despite high potential and utility of this approach, clinical implementation has not yet been achieved due to practical hurdles like speed of data acquisition and lack of optimized computational procedures for extracting clinically actionable information rapidly. The latter problem has been addressed by developing highly efficient ways to process IR imaging data but remains one that has considerable scope for progress. Here, we summarize the major issues and provide practical considerations in implementing a modified Bayesian classification protocol for digital molecular pathology. We hope to familiarize readers with analysis methods in IR imaging data and enable researchers to develop methods that can lead to the use of this promising technique for digital diagnosis of cancer.     

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.     

Evaluation of four-dimensional cone beam computed tomography ventilation images acquired with two different linear accelerators at various gantry speeds using a deformable lung phantom

We evaluated four-dimensional cone beam computed tomography (4D-CBCT) ventilation images (VI_CBCT) acquired with two different linear accelerator systems at various gantry speeds using a deformable lung phantom.The 4D-CT and 4D-CBCT scans were performed using a computed tomography (CT) scanner, an X-ray volume imaging system (Elekta XVI) mounted in Versa HD, and an On-Board Imager (OBI) system mounted in TrueBeam. Intensity-based deformable image registration (DIR) was performed between peak-exhale and peak-inhale images. VI_CBCT- and 4D-CT-based ventilation images (VI_CT) were derived by DIR using two metrics: one based on the Jacobian determinant and one on changes in the Hounsfield unit (HU). Three different DIR regularization values (λ) were used for VI_CBCT. Correlations between the VI_CBCT and VI_CT values were evaluated using voxel-wise Spearman’s rank correlation coefficient (r).In case of both metrics, the Jacobian-based VI_CBCT with a gantry speed of 0.6 deg/sec in Versa HD showed the highest correlation for all the gantry speeds (e.g., λ = 0.05 and r = 0.68). Thus, the r value of the Jacobian-based VI_CBCT was greater or equal to that of the HU-based VI_CBCT. In addition, the ventilation accuracy of VI_CBCT increased at low gantry speeds.Thus, the image quality of VI_CBCT was affected by the change in gantry speed in both the imaging systems. Additionally, DIR regularization considerably influenced VI_CBCT in both the imaging systems. Our results have the potential to assist in designing CBCT protocols, incorporating VI_CBCT imaging into the functional avoidance planning process.     

Quantitative characterization of domes in primary mouse mammary epithelial tumor cell cultures

Summary Dome populations from primary cultures of mouse mammary tumor cells were quantitatively studied in regard to size, distribution, density and the area occupied by light-diffusion photography and image analysis. The effects of fetal bovine serum, insulin and hydrocortisone were analyzed. Quantitative characterization documented dome diameter (mode diameter 0.26 to 0.52 mm), dome area occupied (average 23%, maximum 38.7%), and density (average 78 domes per cm², maximum 117 domes per cm²) for standard culture conditions. Insulin and hydrocortisone had a primary effect on dome density whereas 15% fetal bovine serum had a minimal effect. However, insulin and hydrocortisone had a synergistic optimal effect on dome population. Time-lapse cinematography revealed that the dome population is not static, but a very dynamic one. Domes underwent irregular pulsations of expansion and contraction. Dome enlargement was either by a series of expansions and contractions, by lateral involvement of other cells, or by coalescence of two or more domes. Domes have been considered to be the in vitro counterpart of the in vivo acinus of the mouse mammary gland. However, quantitative dome population characterization has not been available. Dome analysis by light-diffraction photography and image analysis lends itself towards correlative studies of domes and their differentiative products. Supported in part by Public Health Service Contract NO1-CP 61013 within the Virus Cancer Program of the National Cancer Institute and by Public Health Service Training Grant CA05245 from the National Cancer Institute.     

Image interpolation allows accurate quantitative bone morphometry in registered micro-computed tomography scans

Time-lapsed in vivo micro-computed tomography is a powerful tool to analyse longitudinal changes in the bone micro-architecture. Registration can overcome problems associated with spatial misalignment between scans; however, it requires image interpolation which might affect the outcome of a subsequent bone morphometric analysis. The impact of the interpolation error itself, though, has not been quantified to date. Therefore, the purpose of this ex vivo study was to elaborate the effect of different interpolator schemes [nearest neighbour, tri-linear and B-spline (BSP)] on bone morphometric indices. None of the interpolator schemes led to significant differences between interpolated and non-interpolated images, with the lowest interpolation error found for BSPs (1.4%). Furthermore, depending on the interpolator, the processing order of registration, Gaussian filtration and binarisation played a role. Independent from the interpolator, the present findings suggest that the evaluation of bone morphometry should be done with images registered using greyscale information.     

The use of novel phenotyping methods for validation of equine conformation scoring results

In this experiment, which is based on a cohort of 44 Lipizzan mares from the Austrian state stud farm of Piber, we present new statistical techniques for the analysis of shape and equine conformation using image data. In addition, we examined which strategies and procedures of image processing techniques led to a successful interpretation of the traits implemented in horse breeding programs. A total of 246 two-dimensional anatomical and somatometric landmarks were digitized from standardized photographs, and the variation of shape has been analyzed by the use of generalized orthogonal least-squares Procrustes (generalized Procrustes analysis (GPA)) procedures. The resulting shape variables have been regressed on the results from linear type trait classifications. In addition, the rating scores of six conformation classifiers were tested for agreement, yielding an inter-rater correlation (inter-class correlation) ranging from 0.41 to 0.68, respectively, a κ coefficient ranging from 0.16 to 0.53. From the 12 linear type traits assessed on a valuating scale, only the type-related traits (type, breed-type and harmony) revealed significant (P<0.05) results in the regression analysis of shape variables on linear type traits. The other nine traits were characterized by a lower agreement between classifiers and did not result in a significant ‘shape regression’. Finally, the ‘horse shape space’ defined by shape variables resulting from GPA procedures offered the possibility to assist in trait definition and in the evaluation of ratings, and it is an adequate biological and objective scale to human perception of conformation, which is expressed in numerical data only.     

In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

Evaluation of mesh- and binary-based contour propagation methods in 4D thoracic radiotherapy treatments using patient 4D CT images

Based on four dimensional (4D) computed tomography (CT) images, mesh- and binary-based contour propagation algorithms for 4D thoracic radiotherapy treatments were evaluated. Gross tumor volumes (GTVs), lungs, hearts and spinal cords on the CT images at the end-exhale and end-inhale phases for six patients were delineated by the physician. All volumes of interest (VOIs) were automatically propagated from the end-exhale phase to the end-inhale phase using two propagation methods. The propagated VOIs were quantitatively compared with the VOIs contoured at the end-inhale phase by the physician using Dice Similarity Coefficient (DSC), Mean Slicewise Hausdorff Distance (MSHD), Center Of Mass (COM) displacement and volume difference. A two-sided Student’s t test was implemented to examine the significance of the differences between the results obtained from the two algorithms. For GTVs, statistically significant differences between the two algorithms were not observed. For all the other VOIs, the mesh-based method showed higher mean DSCs for the heart, left lung, right lung and spinal cord, lower mean MSHD for the spinal cord, lower mean COM displacement for the heart, and lower mean volume differences for the left lung, right lung and spinal cord with statistically significant differences than the binary-based method. The running time for propagation was approximately 3 s and 3 min for the mesh- and binary-based methods, respectively. Collectively, the mesh-based algorithm provides superiorities in running time and reliability for contour propagation in 4D radiotherapy.     

Structural changes in the innercortex cells of soybean root nodules are induced by short-term exposure to high salt or oxygen concentrations

After a 2 h exposure of intact soybean nodules to high concentrations of NaCl (100mol m_?3) or oxygen (8OkPa O₂), morphometric computations carried out using an image analysis technique on semi-thin sections showed that both treatments induced a decrease in the area of the inner-cortex cells, which were then characterized by a tangential elongation. In contrast, no significant change in area occurred in the middle-cortex cells although their elongation decreased. Electron microscopic observations showed that in the inner-cortex cells changes included the presence of wall infoldings, an enlarged periplasmic space and a lobate nucleus whose chromatin distribution differed from that of the control. Structural changes also occurred in the endoplasmic reticulum, microbodies, mitochondria and plastids. From several of these changes, which are similar to those noted in osmocontractil cells in response to external stimuli, it can be hypothesized that the inner cortex may provide a potential mechanism for the control of oxygen diffusion through the nodules.